HIF-1α and Pro-Inflammatory Signaling Improves the Immunomodulatory Activity of MSC-Derived Extracellular Vesicles

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EV_MSC-T-HIF^c). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EV_MSC-T-HIF^c suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.     

X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.     

Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH,FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.     

Matching ultrasonic transducer using two matching layers where one of them is glue

The aim of this work is to propose an original solution to the acoustic impedance mismatch between a PZT transducer and a water load. The characteristic acoustic impedance of the PZT is around 33 MRayl. Theoretically, a quarter-wave layer with characteristic acoustic impedance equal to 7 MRayl is necessary to match the transducer to water. In practice, it is difficult to find a material with this particular impedance. Two or more quarter-wave layers may also be used. The following proposed solution consists in using two matching layers where one of them is the glue. Moreover, their thicknesses are not equal to a quarter of their individual wavelength. Once the glue is taken as the first matching layer, the specific material of the second layer can easily be found. In this work, the second layer, which is also the front layer, is made of glass. The thickness of each matching layer is then calculated as a function of the characteristic acoustic impedance of the two layers. The influence of the thickness of these layers is discussed. The proposed matching configuration is analyzed and theoretical results have been carefully and successfully compared with measurements.     

Development and characterization of a biocompatible OH-modified copolymer based on polyurethane

Different polyurethanes were synthesized by varying the diol as well as the diisocyanate components and chain extenders. Polyurethanes with OH-groups were obtained by photo- and thermoinitiation of the radical polymerization of hydroxy alkyl acrylates in the presence of the polyurethanes. The polymers were evaluated with respect to their biocompatibility by measuring the cell spreading, the rates of DNA- and protein synthesis and the swelling behaviour. The differences in the surfaces and the bulks between the selected basic polyurethane and the functionalized modification were determined and characterized by XPS, FTIR-ATR and ¹³C-FT-NMR-spectroscopy. The mechanical data of Tecoflex EG 60D® and Pellethane 2363–80AE® were compared with the data of the synthesized polyurethanes.     

Influence of Molybdenum Silicide Additions on High-Temperature Oxidation Resistance of Silicon Nitride Materials

The influence of additions of molybdenum disilicide (MoSi₂) on the microstructure and the mechanical properties of a silicon nitride (Si₃N₄) material, with neodymium oxide (Nd₂O₃) and aluminum nitride (AIN) as sintering aids, was studied. The composites, containing 5, 10, and 17.6 wt% MoSi₂, were fabricated by hot pressing. All materials exhibited a similar phase composition, detected by X-ray diffractometry. Up to MoSi₂ additions of 10 wt%, mechanical properties such as strength, fracture toughness, or creep at 1400°C were not affected significantly, in comparison to that of monolithic Si₃N₄. The oxidation resistance of the composites, in terms of weight gain, degraded. After 1000 h of oxidation at 1400° and 1450°C in air, a greater weight gain (by a factor of approximately three) was obtained, in comparison to that of the material without MoSi₂. Nevertheless, after 1000 h of oxidation, the degradation in strength of the composites was considerably less severe than that of the material without MoSi₂. An additional layer was formed, caused by processes at the surface of the Si₃N₄ material, preventing the formation of pores, cracks, or glassy-phase-rich areas, which are common features of oxidation damage in Si₃N₄ materials. This surface layer, containing Mo₅Si₃ and silicon oxynitride (Si₂ON₂), was the result of reactions between MoSi₂, Si₃N₄, and the oxygen penetrating by diffusion into the material during the hightemperature treatment.     

Surface Composition of Myrmecophilic Plants: Cuticular Wax and Glandular Trichomes on Leaves of <Emphasis Type="Italic">Macaranga tanarius</Emphasis>

Primary plant surfaces, covered with cuticles consisting of cutin and waxes, are important substrates for interaction with insects. The composition of leaf surfaces of the myrmecophilic plant Macaranga tanarius was studied. The prenylated flavanone nymphaeol-C was identified in surface extracts and was localized exclusively in glandular trichomes on the abaxial leaf side. The epidermal pavement cells surrounding these trichomes were covered with a smooth film of epicuticular wax from which few small wax crystals protruded. The epicuticular wax amounted to approximately 8 μg cm⁻², corresponding to 85% of the wax load on the adaxial as well as the abaxial leaf sides. The epicuticular wax mixtures from both leaf surfaces contained more than 70% primary alcohols, 14% fatty acids, 2% aldehydes, and traces of alkyl acetates, with chain lengths ranging from C₂₀ to C₃₈. In contrast, the intracuticular wax layer was largely dominated by triterpenoid alcohols α-amyrin, β-amyrin, and lupeol. Consequently, these characteristic compounds are not available for direct contact with insects on the plant surface.     

Computational Problem Solving in Spatial Substrates -- A Cognitive Systems Engineering Approach

The ability to perform spatial tasks is crucial for everyday life and of great importance to cognitive agents such as humans, animals, and autonomous robots. A common artificial intelligence approach to accomplish spatial tasks is to represent spatial configurations and tasks in form of detailed knowledge about various aspects of space and time. Suitable algorithms then use the representations to compute solutions to spatial problems. In comparison, natural embodied and situated agents often solve spatial tasks without detailed knowledge about geometric, topological, or mechanical laws; they directly relate actions to effects that are due to spatio-temporal affordances in their bodies and environments. Accordingly, we propose a paradigm that makes the spatio-temporal substrate an integral part of the engine that drives spatial problem solving. We argue that spatial and temporal structures in body and environment can substantially support (and even replace) reasoning effort in computational processes: physical manipulation and perception in spatial environments substitute formal computation. While the approach is well known – for example, we employ diagrams as spatial substrate for geometric problem solving and maps for wayfinding – the underlying principle has not been systematically investigated or formally analyzed as a paradigm of cognitive processing. Topology, distance, and orientation constraints are all integrated and interdependent in truly 2- or 3-dimensional space. Exploiting this fact may not only help overcome the need for acquiring detailed knowledge about the interrelationships between different aspects of space; it also can point to a way of avoiding exploding computational complexity that occurs when we deal with these aspects of space in complex real-world scenarios. Our approach employs affordance-based object-level problem solving to complement knowledge-level formal approaches. We will assess strengths and weaknesses of the new cognitive systems paradigm.     

Five-year experience with setup and implementation of an integrated database system for clinical documentation and research

In radiation oncology, where treatment concepts are elaborated in interdisciplinary collaborations, handling distributed, large heterogeneous amounts of data efficiently is very important, yet challenging, for an optimal treatment of the patient as well as for research itself. This becomes a strong focus, as we step into the era of modern personalized medicine, relying on various quantitative data information, thus involving the active contribution of multiple medical specialties. Hence, combining patient data from all involved information systems is inevitable for analyses. Therefore, we introduced a documentation and data management system integrated in the clinical environment for electronic data capture. We discuss our concept and five-year experience of a precise electronic documentation system, with special focus on the challenges we encountered. We specify how such a system can be designed and implemented to plan, tailor and conduct (multicenter) clinical trials, ultimately reaching the best clinical performance, and enhancing interdisciplinary and clinical research.